Regeneration after injury in post-natal organ systems, in many ways, recapitulates developmental processes during embryogenesis. Though many interesting and crucial individual genes that are important for embryogenesis and organogenesis have been discovered over the past decade, the integrated regulation of the process is in many ways unknown (Barinaga, 1994), as are the similarities and differences between embryonic development and regeneration/healing of post-natal cells, tissues and organs. In embryonic mice and man, the various tissue systems develop in parallel and use both inter- and intra-tissue signaling, while the environment around the tissue progresses from one dependent on diffusion of oxygen to one in which oxygen is supplied via the developing vascular system. In the embryo over time, oxygenation to tissues increases as the blood supply is laid down and extended, but this delivery of oxygen is not homogenous throughout any tissue. And though oxygenation becomes richer as the embryo grows, levels of oxygen present in the embryo are generally considered insufficient for normal adult tissue functioning.
Each tissue and organ develops by an exquisitely organized progression in which relatively unspecialized or "undifferentiated" progenitor or stem cells give rise to progeny that ultimately assume distinctive, differentiated identities and functions. Mature tissues and organs are composed of many types of differentiated cells, with each cell type expressing a particular subset of genes that in turn specifies that cell's distinctive structure, specialized function, and capacity to interact with and respond to environmental signals and nutrients. These molecular, structural and functional capacities and properties comprise the cell phenotype. A similar course of coupled cell proliferation and differentiation in the presence of changing local O.sub.2 supply occurs when an injured or degenerating adult tissue undergoes repair and regeneration. The level of oxygen is especially pertinent in many regeneration paradigms in which normal blood supply is reduced or even transiently stopped by trauma or embolic events (myocardial infarction, stroke).
The hypothesis that O.sub.2 levels have significant differential impact on different cell types or states has so far received little explicit attention in the literature, with the exception of formation of the vasculature itself. In particular it is important to note that the vast preponderance of studies of regeneration in vitro are performed in laboratories using room air oxygen levels. In room air, 20-21% of atmospheric gas is oxygen (at sea level depending on humidity), which translates into an oxygen partial pressure of 160 mm Hg [0.21(760 mm Hg)]. The most highly oxygenated tissue in the human body is the arterial blood supply with an oxygen partial pressure of 90 mm Hg. Normal venous oxygenation is 40 mm Hg, and mean tissue oxygen level is 26 mm Hg. However, the vast majority of regeneration research or research on the culture of progenitor cells, stem cells, or differentiating products ignores the importance of oxygenation: The average tissue culture condition is 21% oxygen and 5% carbon dioxide which the remainder being nitrogen.
Herein, the inventors demonstrate that regulated oxygen levels, particularly subatmospheric levels of oxygen (i.e. levels below 21% oxygen and 5% carbon dioxide), can be used to exploit responses of stem and progenitor cells that differ from the response of other cells as a simple and general pathway for their isolation, maintenance, proliferation, enrichment, and/or selective developmental progression and differentiation. This work has important implications for clinical tissue and organ transplantation.
In a time of critical shortages of donor organs, efforts to bring cellular transplantation into the clinical arena are urgently needed (Neelakanta & Csete, 1996). For example, in the case of the liver, a stem cell has not been rigorously identified, and animal models of transplantation of fully-differentiated liver cells (normally quiescent and difficult to force into division experimentally) are not yet successful enough to warrant clinical trials. However, a liver stem cell would represent the ideal cellular transplant because of the potential to regenerate substantial organ function from a tiny rudiment. Thus, there remains a need for methods to identify cells (progenitors and stems) which can be used to regenerate tissue. The present invention is directed at these goals.